Abstract Neuron to (astro)glia communication is essential for functional synaptic transmission and physiology in the CNS. Despite the important modulatory roles of astroglia in synapse function, molecular pathways that regulate the neuron-astroglia functional unit are largely undefined. Exosomes (50-150 nm in diameter), a major type of secreted extracellular vesicles (EVs), are derived from intraluminal vesicles (ILVs) in the early endosomal compartment and are released from cells during endosome maturation. EVs and exosomes secreted from various CNS cell types have emerged as a novel and important intercellular communication pathway in the CNS. In particular, miRNAs (miRs) are often found in exosomes to shuttle between cells for intercellular signaling. Intercellular transfers of miRs have been observed in CNS cells to regulate glutamate transporter function, promote myelination, and maintain brain vascular integrity. Exosomal signaling has also been implicated in pathological conditions of the CNS, including neurological injury, neurodegenerative diseases, and glioblastoma. Despite the strong rigor in prior studies to suggest the importance of the exosomal pathway in CNS cell communication, these studies are largely based on culture models or human CSF samples, exosome signaling in situ in the CNS remains essentially unexplored. In addition, fundamentally important cell biology aspects of this pathway, such as neuronal activity's influence, exosome internalization mechanisms, and downstream regulation in recipient CNS cells also remain unknown. This is particularly important to address as CNS cell types are highly distinct from cancer/immune cells where most of exosome knowledge is currently gained and exosome signaling mechanisms can be very cell-type heterogeneous. Based on our published study and additional preliminary results, we propose the following aims in this project: Aim 1: Determine the effect of neuronal activity on the subcellular localization of ILVs and neuronal exosome secretion; Aim 2: Dissect recognition pathways and entry mechanisms involved in astroglial internalization of neuronal exosomes; Aim 3: Investigate genetic regulation of neuronal exosomal miR-124 in astroglia; We have generated a large amount of preliminary data to support our rationales and to demonstrate feasibility for proposed aims. We will employ mouse genetics, molecular biology, virus injections, various imaging, and biochemical approaches to complete these aims. Outcomes from this project will present in vivo evidence to support a previously unrecognized mode of communication from neurons to glia in the CNS. It will also provide much-needed cell biological knowledge and insights for understanding exosome signaling in neuron to glia communication, especially about miR-124-3p's non-cell autonomous genetic regulation in astroglia following its internalization. As altered neuron to (astro)glia communication is clearly implicated in many neurological diseases, this knowledge and insights can significantly help understand how this pathway is involved in CNS diseases.